Throughout this application, various publications are referred to. These publications, and references included therein, are incorporated herein in their entirety.
In many instances, combination therapies employing two or more therapeutic compounds are required to adequately address different medical conditions and/or effects of a certain disorder under treatment. Thus, β-glycolipides which were previously shown by part of the inventors as having an immuno-modulatory effect may be employed together with various other therapeutic agents, and specifically with antibodies recognizing antigens derived from an immune-system component, to address a broader spectrum of immune-related disorders. Combining at least two immunomodulatory medications safely and effectively improves overall beneficial effect on different immune-related abnormalities.
Immune therapy involves the exposure of components of the immune system to various elements (cytokines, disease, associated antigens and natural metabolites) to combat disease processes in which a dysregulated immune response is thought to play a role. Immune dysregulation is thought to play a major part in the pathogenesis or disease course of a great number of disease processes, including various neoplastic, inflammatory, autoimmune, infectious and genetic entities.
These disorders can be perceived as a dysbalance between pro-inflammatory (Th1) and anti-inflammatory (Th2) cytokines. Both CD4 and CD8 lymphocytes can be typed as either Th1 cells that produce IL-2 and IFNγ, or Th2 cells that produce IL-4, and IL-10. The way the immune system responds to foreign and self antigens, is the result of a balance between the two subtypes of responses [Weiner, H. L., et al. Immunol. Today 18: 335-343 (1997); Adorini, L., et al. Immunol. Today 18:209-211 (1997)]. A Th1 type response is involved in the pathogenesis of several autoimmune and chronic inflammatory disorders such as IBD [Adorini, L., et al. (1997) ibid.; Mizoguchi, A., et al. J. Exp. Med. 183:847-856, (1996)]. Thus experimental autoimmune diseases in humans can be perceived as a dysbalance between pro-inflammatory Th1-type and anti-inflammatory Th2-type cytokines. It has been previously shown, in both animals and humans, that anti-inflammatory cytokines such as IL-10 can down regulate the pro-inflammatory effects of Th1-mediated cytokines, thereby alleviating immune-mediated disorders [Mizoguchi, A., et al. (1996) ibid.; Madsen, K. L., et al. Gastroenterology 113:151-159 (1997); Van Deventer Sander, J., et al. Gastroenterology 113:383-389 (1997)].
In the past few years it has been become increasingly clear that T cells capable of actively suppressing immune responses are thought to be in part responsible for the maintenance of peripheral self tolerance. In healthy rodents and humans, CD4+ T cells constitutively expressing the interleukin IL-2 receptor alpha-chain (CD25) are able to exert such suppressive function in vitro and in vivo [Piccirillo, C. A. and Shevach, E. M. Semin. Immunol. 16:81-88 (2004)]. Immunoregulatory cytokines such as IL-10 or TGF-β may be critical for the suppressive effect of these cells. Regulatory T cells have potential role in human autoimmune or chronic inflammatory diseases and can be used for diagnostic or therapeutic purposes [Frey, O. and Brauer, R. Arch. Immuno. Ther. Exp. (Warsz) 54:33-43 (2006)].
Regulatory T cells (Tregs) are a specialized subpopulation of T cells that act to suppress activation of other immune cells and thereby maintain immune system homeostasis, self-tolerance as well as control excessive response to foreign antigens [Le, N. T. and Chao, N. Bone Marrow Transplant 39:1-9 (2007)]. The two best-characterized subsets are the naturally arising, intrathymic-generated Tregs. (natural Tregs.) and the peripherally generated, inducible Tregs. (inducible Tregs.) [Le (2007) ibid.].
Strategies for therapeutic targeting of regulatory T cells and the effect of regulatory T cells on current immunotherapeutic and vaccine regimens have been recently described [Zou, W. Nat. Rev. Immunol. 6:295-307 (2006)].
Self/non-self discrimination is a complex process that involves maintaining tolerance to auto-antigens while preserving the potential to generate an effective humoral and cellular immune responses against invading pathogens. CD4(+)CD25(+) Treg, have emerged as a dominant T cell population capable of mediating peripheral tolerance to autoantigens, but whose functions have now been extended to regulation of T cell responses directed to foreign antigens [Piccirillo (2004) ibid.].
Autoimmunity results from the dysregulation of the immune system leading to tissue damage. Th1 and Th17 cells are known to be cellular mediators of inflammation in autoimmune diseases. The specific cytokine milieu within the site of inflammation or within secondary lymphatic tissues is important during the priming and effector phases of T cell response. Dendritic cell subsets can change the balance between major players in autoimmunity, namely Th1, Th17 and regulatory T cells. Th17 cells, once thought to only act as pathogenic effectors through production of IL-17, have been shown to have regulatory properties as well with co-production of the anti-inflammatory cytokine IL-10 by a subset now referred to as regulatory Th17 cells [Nikoopour, E. et al. Inflamm. Allergy Drug Targets 7:203-210 (2008)]. IL-17 is important in the induction of autoimmune diseases such as experimental autoimmune encephalomyelitis (EAE) and inflammatory bowel disease (IBD).
Promotion of regulatory T cells is important for the treatment of immune mediated disorders, and for infectious, inflammatory, malignant or any disease in which the immune system plays a deleterious role or have a role in their pathogenesis.
WO 2007/060652, which is a previous publication by part of the present inventors, discloses the use of beta-glycolipides as an immuno-modulatory compound in the treatment of immune-related disorders. More specifically, this publication showed that a particular mixture of β-lactosyl-ceramide with β-glucosylceramide is a powerful immuno-modulating medicament useful in the treatment of immune-related disorders.
WO 2005/048935, which is another publication by part of the present inventors, describes the immunomodulatory effect of an anti CD3 antibody on autoimmune disorders. More specifically, this publication shows that oral and mucosal administration of anti-CD3 antibody suppresses experimental allergic encephalomyelitis (EAE, an animal model of multiple sclerosis (MS), delays allograft rejection in a dose-dependent fashion, reduces the severity of arthritis and prevents the onset of diabetes in NOD mouse model.
There is growing evidence that there may be a link between inflammation and the pathogenesis of Type 2 diabetes [Shoelson, S. E. et al. The Journal of clinical investigation 116:1793-1801 (2006)]. This evolving concept which suggests that insulin resistance and type 2 diabetes may have an immune component provides a new avenue to investigate immunotherapeutic approaches to both understand the pathogenesis of type 2 diabetes and to develop new treatments for the disease. Regulatory T cells have been implicated as playing in a key role in classic autoimmune diseases in which deficiencies have been identified and strategies to induce Tregs for treatment of these diseases are being actively pursued [Baecher-Allan, C. and Hafler, D. A. Immunological reviews 212:203-216 (2006); Belkaid, Y. Nature reviews 7:875-888 (2007); Tang, Q. and Bluestone, J. A. Immunological reviews 212: 217-237 (2006)]. Although Tregs have been extensively investigated in animals models and human subjects with type 1 diabetes [Tang, Q. and Bluestone, J. A. Immunological reviews 212: 217-237 (2006); Bluestone, J. A. and Tang, Q. Current opinion in immunology 17:638-642 (2005); Chatenoud, L. and Bach, J. F. International reviews of immunology 24:247-267 (2005); Randolph, D. A. and Fathman, Annual review of medicine 57:381-402 (2006); von Herrath, M. et al. Nature reviews 7:988-994 (2007)] their potential role in type 2 diabetes has not been well explored and is not fully understood [Yeh, S. H. et al. Diabetes care 30:716-718 (2007)].
The present invention now shows a surprising and clear synergistic effect of β-glycolipides, specifically, β-glucosylceramide (GC), and anti-CD3 antibody on immune-related disorders. More specifically, the invention demonstrates that the induction of regulatory T cells by oral administration of a combination of anti-CD3 plus β-glycosphingolipid alleviates the metabolic syndrome in ob/ob mice in a TGF-β dependent manner. More particularly, a marked decrease in pancreatic islet cell hyperplasia and decreased accumulation of fat in the liver accompanied by lower blood glucose and liver enzymes was demonstrated in animals treated with the combined composition. Moreover, the invention shows that the combined composition of GC and anti-CD3 leads to elevation in serum levels of insulin. These effects were mediated by the induction of CD4+LAP+ Tregs by oral anti-CD3 plus GC. Adoptive transfer of CD4+LAP+ Tregs ameliorated the metabolic changes and pathologic abnormalities in a TGF-β dependent fashion. The results of the present invention suggest that immune abnormalities may play an important role in type 2 diabetes and identify a unique immunologic approach for treatment by induction of regulatory T cells.
Thus, a combination of these two compounds, specifically, GC, and anti-CD3 antibody promote different types of regulatory cells in the bowel or systemically. Additionally, this combination activates antigen presentation by promoting APC, including but not limited to dendritic cells. Both compounds in a synergistic combination alter the secretion of chemokines or cytokines that may together enhance the function of any component of the immune system. The invention further shows a remarkable anti-inflammatory effect of the combined composition on a tissue related to the treated disorder, e.g., fat tissue. More particularly, using the ob/ob model, the combined composition of the invention demonstrated suppression of pro-inflammatory cytokine expression, followed by elevation in anti-inflammatory cytokines production in adipocytes. Furthermore, the combined composition of the invention led to a marked decreased inflammatory cell infiltration to the fat tissue of the treated subject.
Overall, these two compounds in combination exert a synergistic effect and enhance the cross talk between different regulatory T cells, effector cells, and other component of the immune system.
Without being bound by any theory, the inventors speculate and show that the combination of these two compounds, antibody and glycosphingolipid, lead to this synergistic effect by activation of antigen presenting cells, different type of T regulatory cells, adipocytes cells or any other immune-cells. Such activation induces the secretion of different cytokines or chemokines and thereby promotes specific cell-cell interaction and induction of specific signal transduction pathways, leading to modulation of the Th1-Th2, Th3 cell balance in each immune-mediated disorder.
The invention therefore provides an oral or mucosal immuno-modulatory composition combining β-glycolipides, specifically, GC, and antibodies specific for CD3 for treating immune-related disorders.
Another object of the invention is to provide methods and kits for treating immune-related disorders using the combined composition of the invention.
These and other objects of the invention will become clearer as the description proceeds.